The present invention relates to methods for quantitative and qualitative analyses of phospholipids. Specifically, the present invention provides methods of using one-dimensional thin layer chromatography to detect and quantify phospholipids. More specifically, the present invention provides one-dimensional thin layer chromatography methods used to detect and quantify the phospholipid content of mammalian tissues.
The specification that follows will be more easily understood by reference to the following definitions. Ordinary terms have been used in manners that preserve their traditional meanings. However, there are numerous references to technical aspects of chromatography that require an understanding of specific terms. While these definitions do not deviate substantially for the meanings accepted by those of ordinary skill in the chemical arts, there may be multiple interpretations of the terms used herein. It such cases the following definition of terms prevails.
Bioprosthetic heart valves (BPHV) have been used since the early 1970s as replacements for diseased human cardiac valves. Originally, the reduced thrombogenicity associated with BPHV made them attractive alternatives to mechanical heart valves. However, BPHV fashioned from bovine pericardium and porcine aortic valves are susceptible to dystrophic calcification. Calcification is associated with approximately 40 to 50 percent of all BPHV failures necessitating re-operation and valve replacement in 10% to 20% of all adult recipients. The calcification rate in children, young adults and acute pathologic conditions is greatly accelerated, consequently, the use of BPHV is limited in these patients.
Recent studies have demonstrated that tissue lipid extraction can significantly reduce calcification, and mineralization generally, in gluteraldehyde cross linked bovine pericardial tissues. This had led some authorities to conclude that lipids act as initiators and/or promoters of tissue mineralization. Therefore, various techniques have been developed to remove lipids from BPHVs prior to implantation. However, in order to monitor and fully understand the role lipids play in minerization of preserved tissues, it is necessary to quantify and qualitate tissue lipid content before, during and after processing. This requires a thorough understanding of lipid chemistry and the problems associated with lipid analytical methods.
Lipids are biological molecules (biomolecules) that are insoluble in water, soluble in organic solvents and are essential components of the plasma membranes that envelop and compartmentalize all living cells. The lipid content of living membranes regulate molecular entry and egress at the cellular and sub-cellular levels. They are found in cell membranes and sub-cellular organelles such as mitochondria, chloroplasts, Golgi bodies, the endoplasmic reticulum and the cell nucleus. In addition to their role as structural components of membranes, lipids serve as energy reserves (triacetylglycerol, also known as neutral fats) and participate in cellular recognition and cell signaling.
There are five major categories of lipids found in biological systems: fatty acids, triacylglycerols, sterols, glycerophopholipids, and sphingolipids. Fatty acids rarely occur in un-complexed, or free forms, in nature. Rather, fatty acids are components of other lipids such as glycerophospholipids, triacylglycerols, and sphingophospholipids. Triacyiglycerols are non-polar (uncharged, hence xe2x80x9cneutral fatsxe2x80x9d) fatty acid triesters of glycerol that are synthesized and stored in adipocytes. Adipocytes are xe2x80x9cfat cellsxe2x80x9d that make up the fatty, or adipose, tissue abundant in the subcutaneous tissues of animals that serve as stored energy reserve and provide thermal insulation. Sterols include cholesterol, which are major components of animal cellular and sub-cellular membranes. Sterols occur in much lower concentration in plants and have not been identified in bacterial (prokaryotic) cell membranes. Moreover, cholesterol is an essential precursor to steroid hormones.
Glycerophospholipids (phosphoglycerides) are the most common lipids associated with cell membranes. The simplest phosphoglyceride is phosphatidic acid (A) which is relatively scarce in cell membranes; however, phosphatidic acid can serve as a precursor for all major phosphoglycerides including phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG), phosphatidylethanolamine (PE) and diphosphatidylglycerol (also known as cardiolipin and found primarily in mitochondrial membranes). Biochemistry Voet and Voet; Lipid Metabolism; pages 663-726; John Wiley and Sons, New York, 2000. Sphingophospholipids include sphingomyelins (SM) and are a principle component of nerve cell myelin sheaths. Sphigophospholipids (SP) have similar conformations and charge distribution to glycerophospholipids and include the phosphorylated head group associated with glycerophospholipids. Consequently, there is considerable chemical similarity between glycerophospholipids and sphigophospholipids; as a result they will be referred to herein collectively as xe2x80x9cphospholipidsxe2x80x9d for convenience.
Phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol, cardiolipin and sphingophospholipids are generally the most abundant phospholipids found in pericardial tissues. It has been reported that phosphatidylserine, cardiolipin and phosphatidylinositol play significant roles in mineralization (D)alas, E. Ioannou, P. V. and P. G. Koutsoukos, In Vitro Calcification: Effect of Molecular Variables of the Phospholipid Molecule. 1990. American Chemical Society. Vol 6:3 535-538). Therefore, it is essential that any analytical method used to assess tissue lipid content have equal sensitivity and specificity for all phospholipids. Phospholipids possess many chemical similarities that make their physical separation from tissue extracts challenging. One of the most effective techniques for separating complex mixtures of large organic compounds, including phospholipids, is chromatography.
Mikhail Semenovich Tswett (xe2x80x9cTswettxe2x80x9d), a Russian botanist, developed chromatography (from the Latin for color writing) shortly after the turn of the twentieth century. Tswett was studying plant pigments and developed the technique of column adsorption chromatography by passing plant extracts through glass columns packed with calcium carbonate (chalk). The different pigments (each a separate organic compound) separated into discrete bands within the packed calcium carbonate column. The subtle differences in molecular weight, shape and charge between the pigments determined the rate that the plant pigment mixture (liquid or mobile phase) moved through the calcium carbonate column (solid support, or stationary phase). Today there are numerous variations on Tswett""s original column chromatography procedure that are based on the general principle described above. These include gas chromatography (or more specifically gas-liquid partition chromatography-GC and GLC), high-pressure liquid chromatography (HPLC), paper chromatography and thin layer chromatography (TLC).
In gas chromatography the sample containing the mixture to be separated is heated to temperatures needed to vaporize the mixture""s components. An inert gas such as nitrogen or helium carries the vaporized mixture (the mobile phase) into a column. The column is packed with a finely divide inert solid that is coated with another liquid of low volatility (the stationary phase). The vaporized compounds in the mobile phase move through the column at different rates due to the compounds"" different relative solubilities in the stationary phase (liquid) and the gas phase (a phenomenon referred to as partitioning). Gas-liquid chromatography is one of the most commonly used analytical techniques; however, it cannot separate complex mixtures of phospholipids.
High-pressure liquid chromatography is a superb analytical tool that relies on the basic principles of column chromatography. Samples are dissolved in a suitable solvent mixture (the mobile phase) and the passed through extremely high surface area capillary silanized silica columns (stationary phase) under tremendous pressure. The components of the mobile phase (the solvent mixture) change over time forming a gradient. Samples elude through the gradient at different intervals based on their charge, molecular weight and relative solubility in the solvent gradient. Various forms of detectors are placed at the end of the column to record each compound""s passing.
Early BPLC columns operated within a narrow pH range and were thus not versatile enough to separate complex lipid mixtures. Moreover, ultraviolet light detection systems were notoriously insensitive and not suited for lipid quantitation. However, recent improvements in derivatized silanized silica columns have resulted in stable supports that operate in a broad pH range (1-12) making them more useful for lipid analysis and laser fight scattering detection systems have significantly improved HPLC lipid quantitation sensitivity.
However, BPLC still presents two major drawbacks. Fist, HPLC is a linear system. One sample is injected at a time, multiple sample analysis remains tedious and time consuming even using auto-injectors. Moreover, HPLC is expensive and complex to operate. Hundreds of thousands of dollars are required to properly equip a fully functional HPLC laboratory and train personnel. If multiple BPLC systems are added to expedite sample processing, the costs go up considerably. Consequently, HPLC facilities that can rapidly process complex lipid containing samples are limited and the cost per sample is extremely high.
Thin layer and paper chromatographies are based on similar principles. A sample containing the compounds to be separated is dissolved in a suitable extraction solvent and a small aliquot is placed near the edge at the support""s base. After the sample spot dries, the support is placed in a container (developing chamber) with sufficient elution solvent to come to a level below the spot. The solvent migrates up the support carrying the compounds in the sample with it at different rates. This procedure is extremely versatile, inexpensive to perform and a number of samples and standards can be resolved simultaneously. When the samples are run in one direction, i.e. up the support, the procedure is referred to as one-dimensional chromatography. This is by far the easiest chromatography method to perform and provides the highest sample throughput.
However, molecules having a high degree of similarity in molecular weight, charge and molecular shape, such as phospholipids, could not be separated using one-dimensional chromatography and were generally not compatible with the cellulose fiber matrix used in paper chromatography. Consequently, a variation on one-dimensional TLC known as two-dimensional TLC was developed. Samples are loaded onto the support and resolved as described above for one-dimensional TLC. After the samples have resolved, the TLC plate is turned 90 degrees and placed in another developing chamber containing a different elution solvent. After the samples have resolved in the second direction, the spots are further separated making the identification of complex, highly similar compounds possible. However, two-dimensional TLC is time consuming and requires that a single TLC plate be resolved twice. This considerably limits the number of samples and standards that can be run simultaneously.
Recently, E. A. Dugan reported the separation of PG, PE, PS, PI PC and SM using one-dimensional thin-layer chromatography (the xe2x80x9cDugan methodxe2x80x9d) (Dugan, E. A., Analysis of Phospholipids by One-Dimensional Thin-Layer Chromatography. 1985. Liquid Chromatography, Volume 3 Number 2). However, the Dugan method does not provide the degree of separation and reproducibility necessary for precise quantitation of phospholipid samples. Moreover, the Dugan method was particularly insensitive for the separation of PC and SM, and PC was not adequately resolved using the methods disclosed therein.
Therefore, there remains a need for an inexpensive, high through-put, accurate analytical process suitable for the simultaneous qualitative and quantitative analysis of lipids in extracted tissue samples.
It is an object of the present invention to provide a rapid, cost effective and reproducible quantitative method for determining the phospholipid content of a sample.
It is another object of the present invention to provide a rapid, cost effective and reproducible quantitative method for determining the phospholipid content of tissue extracts.
It is another object of the present invention to provide a rapid, cost effective and reproducible quantitative method for determining the phospholipid content of tissue extracts using one-dimension thin layer chromatography.
It is an object of the present invention to provide a rapid, cost effective and reproducible qualitative method for determining the phospholipid content of a sample.
It is another object of the present invention to provide a rapid, cost effective and reproducible qualitative method for determining the phospholipid content of tissue extracts.
It is another object of the present invention to provide a rapid, cost effective and reproducible qualitative method for determining the phospholipid content of tissue extracts using one-dimension thin layer chromatography.
It is yet another object of the present invention to provide a rapid, cost effective and reproducible method for the simultaneous qualitative and quantitative determining the phospholipid content of tissue extracts using one-dimension thin layer chromatography.
The present invention generally provides methods for the qualitative and quantitative analysis of biological samples containing mixtures of lipids, specifically mixtures containing neutral lipids and phospholipids. Samples are extracted using organic solvents (extraction solvent), partitioned, dried and the lipid fraction redissolved in an extraction solvent as known to those skilled in the art. The samples are then applied to activated thin layer chromatography plates. Next, the plates are equlibrated and resolved using the elution solvent of the present invention. The resolved plates are stained with a fluorescent dye and then scanned using fluorescent imaging and quantified using appropriate software.
In one embodiment the present invention is used to determine the lipid content of mammalian cardiac tissues. Samples are extracted using a methanol-chloroform solution and processed using silica gel coated thin layer chromatography plates. The lipids are developed using a chloroform, methanol, acetic acid, aqueous potassium chloride solution and detected using primulin and a Storm(copyright) imaging unit.
The method of the present invention provides a technique to qualitate and quantify phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidic acid, phosphatidylserine, sphingomyelin and phosphatidylglycerol present in a single tissue extract. Neutral lipids do not separate from the elution solvent of the present invention and migrate out of the mixture with the solvent front (the leading edge of the solvent as it migrates up the chromatography plate). Consequently, the presence of neutral lipids in tissue samples does not interfere with phospholipid detection quantitation.
Other objects, features and advantages of the present invention will be apparent to those skilled in the art from a consideration of the following detailed description of preferred exemplary embodiments thereof taken in conjunction with the Figure which will first be briefly described.